1. Field of the Invention
The present invention relates to capillary gel electrophoresis, and more particularly to refilling capillaries using a polymerized gel.
2. Description of Related Art
Electrophoresis is one of the most widely used separation techniques in the biologically related sciences. Molecular species such as peptides, proteins, and oligonucleotides (analytes) are separated by causing them to migrate at different rates in a separation medium under the influence of an electric field. The separation medium can be a buffer solution, or a low to moderate concentration of an appropriate gelling agent such as agarose or polyacrylamide. When gel separation medium is used, separation of analytes is partly based on their molecular sizes as the analytes are sieved by the gel matrix. Smaller molecules move relatively more quickly than larger ones through a gel of a given pore size which depends in part on the concentration of the polymer in the gel.
U.S. Pat. Nos. 4,855,706 and 4,865,707 to Barry L. Karger and Aharons Cohen describe gel compositions suitable for capillary electrophoresis. A fused silica capillary having inner diameter in the order of 75 xcexcm is first filled with a mixture of acrylamide monomer and other ingredients and polymerization is then allowed to go to completion in the capillary. The time taken to complete polymerization is a minimum of one hour. The polymerized gel has a limited storage life. Also, performance of the gel deteriorates after a period of use. This may be due to gradual accumulation of macromolecules in the gel matrix after repeated runs. The applied electric field may cause disintegration of the polymer material after repeated use. In the past, the gel-filled capillary columns have to be discarded after their useful life.
The gel columns have heretofore been used in laboratory set-ups involving many manual steps, e.g. placement of buffer containers with respect to the ends of the gel column, etc. Also spent gel column has to be manually replaced by a new gel column. To take advantage of automation in carrying out electrophoresis, due considerations should be given to eliminate as many of the manual steps in the design of an automated electrophoresis system.
The present invention is directed to filling an internally coated capillary using a gel which is polymerized before filling the capillary. The internal coating on the capillary walls prevents bonding of the gel to the capillary walls. The gel is of a composition which allows it to fill the capillary in its polymerized state without damage to the gel and to be removed from the capillary after the gel has expended its useful life. The capillary can then be refilled with fresh gel. This can be handled automatically by an automated capillary electrophoresis system.
In the illustrated embodiment, the formulation of the gel comprises up to 6% of acrylamide monomer and buffer. In addition, optional amounts of crosslinker, catalyst, initiator, urea, and other additives may be added to adjust for the desired pore size, separation efficiency and life of the gel. The composition results in a gel of a consistency which can be forced into and out of the internally coated capillary without damaging the gel.